The present invention pertains in general to methods and hybrid promoters for controlling transcription of exogenous genes and in particular to methods and hybrid promoters for controlling transcription of exogenous genes in yeast.
A number of microorganisms, including bacteria and yeasts, are useful for the manufacture of substantial quantities of biological products the synthesis of which is directed by genes which are foreign to the microorganisms employed ("exogenous genes"). In particular, there is considerable interest in the production of exogenous gene products in yeast cells because yeast cells are able to attach carbohydrates (i.e., to glycosylate) proteins for the expression of active glycoprotein gene products; they are able to process protein gene products in a way which permits secretion of the gene products; and they are able to present surface antigens, e.g., hepatitis B surface antigens (HBsAg), in a way which enhances their immunogenicity. Moreover, yeasts appear on the list of organisms generally regarded as safe (GRAS) and extensive commercial fermen technology has been developed for their fermentation in the baking and brewing industries.
In general, genes encoding protein products are segments of double-stranded deoxyribonucleic acid (DNA), one strand of which is transcribed into a single strand of messenger ribonucleic acid (mRNA) through the action of RNA polymerase II. The mRNA is translated into proteins which may be further processed for secretion and glycosylation. In order for the gene to be transcribed, an initiation complex must be formed between RNA polymerase and DNA in the region near the first nucleotide base to be transcribed, i.e., at the "upstream" end of the gene. The mRNA is then synthesized as the RNA polymerase moves downstream along the gene until it reaches a DNA site, called a terminator, at which transcription ceases. The entire 5'-flanking sequence of DNA which is upstream from the gene and which is competent to promote transcription initiation complex is known as a promoter. Within the promoter and near (generally 25 to 150 base pairs upstream of) the point at which the first nucleotide is incorporated into mRNA (the start point of the transcription initiation complex), the sequence of seven base pairs known as the TATA box homology is found. The TATA box homology is apparently required for fixing transcription initiation at a specific distance downstream. The TATA box homology has the consensus sequence ##STR1## although minor variations of this sequence have been observed.
In yeast, certain sites called upstream regulatory sequences or upstream activation sequences (UASs) have been located at sites hundreds of nucleotides upstream of the region of transcription initiation. These UASs regulate transcription in response to particular physiological signals. Guarente, Cell, 36: 799-800 (1984).
One UAS, called UAS.sub.G for upstream activating sequence-galactose, is found between genes coding for two enzymes involved in the metabolism of galactose, the GAL 1 gene coding for galactokinase and the GAL 10 gene coding for UDP-galactose epimerase. The major transcription initiation sites of the GAL 1 and GAL 10 genes are separated by 606 base pairs of DNA on yeast chromosome II. Johnston, et al., Mol.Cell.Biol., 4: 1440-1448 (1984).
The GAL 1 and GAL 10 genes are transcribed in opposite directions (divergently transcribed) from an intergenic region. The start point for the GAL 1 gene is downstream of a TATA box on a strand opposite the strand on which the start point for the GAL 10 gene is located downstream of a second TATA box. DNA sequences within this GAL 1/GAL 10 intergenic region mediates the regulation of both genes by carbon source. Yocum, et al., Mol.Cell.Biol., 4: 1985-1998 (1984).
These genes are expressed at a low, basal level when the yeast cells containing them are grown in non-fermentable carbon sources, such as lactate, glycerol or ethanol. Transcription of the genes is induced 1,000-fold by growth in galactose On the other hand, these genes are subject to catabolite repression in that cells growing in glucose are not fully inducible by galactose.
The UAS.sub.G is controlled by other genes which activate or repress transcription. A GAL 80 gene encodes a negative regulator which is hypothesized to function by binding to and inactivating a protein produced by a GAL 4 gene. In the presence of galactose, the GAL 80 regulator is non-functional and the GAL 4 protein activates transcription. Oshima, "Regulatory Circuits for Gene Expression: The Metabolism of Galactose and Phosphate", in The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, Strathern, et al., eds., Cold Spring Harbor Laboratory, N.Y., 159-180 (1982). Several genes, including a GAL 82 gene and a GAL 83 gene, are involved in glucose repression, but their mode of action has not been well defined. Matsumoto, et al., J.Bacteriol., 153: 1405-1414 (1983).
The properties of UASs may be examined by the endonuclease-mediated substitution of a UAS from a first promoter for a different UAS of a second promoter or by endonuclease-mediated insertion of a UAS in a functional promoter (i.e., either substitution or insertion of an extrinsic UAS). Guarente, et al., Proc.Natl.Acad.Sci. (U.S.A.) 7410-7414 (1982); Fried, et al., Mol.Cell.Biol., 5: 99-108 (1985). Substitutions of and insertions of UAS.sub.G include the substitution of UAS.sub.G for the UAS of an inducible gene (CYC 1) by Guarente, et al., supra, which is reported as indicating that the UAS.sub.G employed does not appear to contain sequences mediating glucose repression despite conferring galactose inducibility, and the insertion of the same UAS.sub.G in constitutive promoters (ribosomal proteins tcm 1 and cyh 2) by Fried, et al., supra, which is reported as conferring both galactose inducibility and glucose repressibility.
The activity of a promoter or of a UAS/promoter hybrid (a "hybrid promoter") is commonly monitored by assaying for the level of a specific protein which is the ultimate product of the gene regulated by the promoter. UASs may be placed at various distances from the site of transcription initiation or may be inverted [Fried, et al., supra; Guarente, et al., Cell, 36: 503-511 (1984)], and functional UASs may constitute sub-fragments of the initial DNA segment used [West, et al., Mol.Cell.Biol., 4: 2467-2478 (1984)]. The combination of UAS/promoter/test gene is generally assembled within a yeast-bacterial shuttle vector.
Plasmids are circular, double-stranded DNA structures which replicate independently of chromosomal DNA. Yeast-bacterial shuttle vectors contain a sequence of DNA, including a DNA replication initiation site such as the so-called 2.mu. origin of replication, recognized by the replication enzymes and factors of yeast cells. Yeast-bacterial shuttle vectors also contain an origin of replication from a bacterial plasmid including an initiation site recognized by the replication enzymes and factors of bacterial cells. Beggs, Nature, 275: 104-109 (1979); Stinchcomb, et al., Proc.Natl.Acad.Sci. (U.S.A.), 77: 4559-4563 (1980). These shuttle vectors are able to replicate in and may be selected in and recovered from both the bacterium Escherichia coli (E.coli), where it is convenient to construct and multiply (amplify) the plasmids, and the yeast Saccharomyces cerevisiae (S.cerevisiae), in which the plasmid may be used as a transformation vector to obtain expression of foreign DNA. Stinchcomb, et al., Nature, 282: 39-43 (1979); Kingsman, et al., Gene, 7: 141-153 (1979); and Tschumper, et. al., Gene, 10: 157-166 (1980).
Bacterial-yeast shuttle vectors may be practically employed in the transformation of yeast cells. One such vector, designated YRp7, contains a chromosomal ARS sequence which allows autonomous replication, as well as the TRP1 gene, which codes for an enzyme essential for the production of tryptophan called N-(5'-phosphoribosyl) anthranilate isomerase. Therefore, the TRP1 gene may be used as a marker to select for the presence of YRp7 within a yeast cell, such as one having the genotype trpl, which is otherwise incapable of manufacturing the essential amino acid tryptophan. Struhl, et al., Proc.Natl.Acad.Sci. (U.S.A.), 76: 1035-1039 (1979).
Studies of the gene CYC 1, the product of which is iso-1-cytochrome c, demonstrate the importance of proper termination of transcription. Improper transcription termination results in continuation of transcription into a neighboring gene. Zaret, et al., Cell, 28: 563-573 (1982). Efficient transcription of yeast DNA into mRNA appears to be dependent upon the presence of a site for addition of a tail containing adenine-containing nucleotides, called a poly-A tail, or a transcription termination sequence or both at or near the downstream (3') end of a polypeptide coding region of a gene.
Such bacterial-yeast hybrid vectors, often referred to as shuttle vectors, may be used to obtain expression of genes from other organisms (exogenous genes) in yeast cells. Heptatitis B surface antigen (HBsAg) has been produced by means of a 3-phosphoglycerate kinase (PGK) promoter (Hitzeman, et al., European Patent Application No. 73657), the arg 3 promoter (Cabezon, et al., European Patent Application No. 106828), a promoter from an alcohol dehydrogenase (ADH 1) gene (Rutter, et al., European Patent Application No. 72318), and a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter [Bitter, et al., Gene, 32: 263-274 (1984)]. A different GPD promoter has been used to obtain expression of thaumatin-like proteins and chymosin-like proteins. Edens, et al., European Patent Application No. 129268. Bovine calf prochymosin, bovine growth hormone, human leukocyte interferon, renin and prorenin may be expressed in yeast by fusion with a GAL 1 promoter. Strausberg, et al., European Patent Application No. 128743; Botstein, et al., U.K. Pataent Application No. 2137208A.
Yeast genes encoding glycolytic enzymes are expressed at high levels. Yeast expression vectors employing glycolytic enzyme promoters such as the ADH promoter [Hitzeman, et al., Nucleic Acids Res., 10: 7791-7808 (1982)] and the promoter for the phosphoglycerate kinase (PGK) gene [Derynck, et al., Nucleic Acids Res., 11: 1819-1837 (1983)] may be constructed.
In particular, the glycolytic enzyme GPD is a promising promoter for use in the expression of exogenous genes. GPD accounts for up to 5% of the dry weight of commercial baker's yeast [Krebs, J.Biol.Chem., 200: 471-478 (1953)] and the mRNA which encodes this enzyme represents 2-5% of the total yeast poly-A-containing mRNA [Holland, et al., Biochemistry, 17: 4900-4907 (1978)]. S. cerevisiae contains three non-tandemly repeated structural genes for GPD, all of which are transcribed in vegetatively growing yeast. Holland, et al., J.Biol.Chem., 258: 5291-5299 (1983); Musti, et al., Gene, 25: 133-143 (1983). The product of one of the three GPD genes, that encoded by the gene on PGAP 491, accounts for most of the cellular GPD enzyme. Jones, et al., FEBS Lett., 22:185-189 (1972); Holland, et al., J.Biol.Chem., 258: 5291-5299 (1983). Thus, the GPD promoter of this gene is expected to be highly useful in the production of large quantities of exogenous gene products.
Despite the advantages of yeast as a host for exogenous genes, some exogenous gene products, such as human immune interferon (IFN-.gamma.), are toxic to yeast. As a result, plasmids constitutively expressing IFN-.gamma. are unstable and it is difficult to obtain high density cultures of yeast cells producing IFN-.gamma.. Therefore, it is desirable to have some means for turning off (repressing) production of exogenous gene products until a high-density culture is obtained, as well as a means for turning on (inducing) production of exogenous gene products during a harvesting period.